A field evaluation of a new porcine circovirus type 2d and Mycoplasma hyopneumoniae bivalent vaccine in herds suffering from subclinical PCV2d infection and enzootic pneumonia

Abstract Background This field efficacy study was designed to determine the efficacy of a new bivalent vaccine containing porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae at three independent pig farms. Methods Three pig farms were selected based on their history of subclinical PCV2 infection and enzootic pneumonia. Each farm housed a total of 40, 18‐day‐old pigs that were randomly allocated to 1 of 2 treatment groups. Pigs were administered a 2.0 mL dose of the bivalent vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate‐buffered saline at the same age. Results Clinically, the average daily weight gain of vaccinated groups was significantly higher (p < 0.05) than those of unvaccinated animals during the growing (70–112 days of age), finishing (112–175 days of age) and overall (3–175 days of age) stages of production. Vaccinated animals elicited neutralizing anti‐PCV2 antibodies and PCV2d‐specific interferon‐γ secreting cells (IFN‐γ‐SC), which reduced the amount of PCV2d genomic copies in blood and reduced lymphoid lesions severity when compared with unvaccinated animals. Similarly, vaccinated animals elicited M. hyopneumoniae‐specific IFN‐γ‐SC, which reduced the amount of M. hyopneumoniae in the larynx and reduced lung lesions severity. Conclusions The result of the field trial demonstrated that the bivalent vaccine was efficacious in the protection of swine herds suffering from subclinical PCV2d infection and enzootic pneumonia.


INTRODUCTION
Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae are some of the most economically important swine disease pathogens (Chae, 2016;Segalés, Allan et al., 2005).PCV2 is the principal etiologic agent of porcine circovirus-associated disease (PCVAD), which was originally described as post-weaning multisystemic wasting syndrome (Segalés, Allan et al., 2005).Since the introduction of PCV2 vaccines in 2007, the clinical forms of PCVAD have become less common in the field, whereas subclinical PCV2 infection has become the prevalent form of PCV2 disease (Segalés, 2012).Subclinical infection of pigs with PCV2 resulted in growth performance reduction and an increased susceptibility to other co-infections (Alarcon et al., 2013).M. hyopneumoniae is the primary etiologic agent of enzootic pneumonia, a chronic respiratory disease characterized by a dry cough, growth retardation and reduced feed efficiency.Enzootic pneumonia causes considerable economic loss in all areas where pigs are raised (Maes et al., 1996).
The majority of current commercially available PCV2 vaccines were developed between 1999 and 2005.During this period, PCV2a was the predominant PCV2 field strain, and little was known about additional PCV2 genotypes and their importance.Therefore, the majority of commercially available PCV2 vaccines are based on PCV2a and/or the PCV2b that followed in emergence (Chae, 2012).In 2010, a novel mutation was first reported in China and designated as mutant PCV2b and was later identified in the United States and Korea in 2014 (Guo et al., 2012;Seo et al., 2014;Xiao et al., 2012).This mutant was later renamed as PCV2d.The main evolutionary changes in the genotypes of PCV2 field strains over time occurred between the predominant PCV2a to PCV2b and again from PCV2b to PCV2d (Franzo & Segales, 2018).
PCV2d has replaced PCV2a and PCV2b as the predominant genotype in essentially all global pig populations throughout Asia, North and South America, and Europe (Franzo et al., 2016;Xiao et al., 2015).
Taking current field conditions into account, bivalent vaccination with PCV2d and M. hyopneumoniae is a valuable strategic tool for controlling porcine respiratory disease complex by these two pathogens.A novel bivalent vaccine containing PCV2d and M. hyopneumoniae antigen was recently introduced into the swine commercial market (Ham et al., 2023).The objective of this study was to evaluate the efficacy of this new bivalent vaccine, with a focus on growth performance in swine herds suffering from subclinical PCV2d infection and enzootic pneumonia.

Farm history
The clinical field trial was conducted on three farms.Farms were labelled 'A, B and C' and were 350-, 360-and 400-sow (respectively) farrow-to-finish swine operations with an all-in-all-out production system.Sows from these three farms were not immunized for either PCV2 or M. hyopneumoniae.The porcine reproductive and respiratory syndrome virus (PRRSV) status was stable as active PRRSV was undetected in circulation.Sows and piglets were not immunized against PRRSV, whereas all piglets received vaccinations for PCV2 and M.
hyopneumoniae at 3 weeks of age.
Each farm consistently experienced respiratory distress as indicated by poor growth rate in the late post-weaning and growing stages of production.Clinical signs first appeared at approximately 8-11 weeks of age and reached peak mortality (farm A = approximately 3%-5%, farm B = 3%-4% and farm C = 3%-6%) between 10 and 15 weeks of age.
Three farms were selected based on their active status of containing subclinical PCV2 infection and enzootic pneumonia.PCV2d was detected in serum from three pigs within each of these three farms, where log 10 DNA copies/mL ranged from 2.44 to 3.42.These values were large enough to diagnose the pigs with subclinical PCV2 infection (Segalés, Allan et al., 2005).M. hyopneumoniae serology was positive in serum samples from pigs aged between 7 and 15 weeks.A lung examination was performed at the slaughterhouse, which confirmed that 40% of these 30 seropositive pigs had mycoplasmal pneumonia lesions.

Field trial design
The results of this field study are intended for registration and therefore all procedures strictly adhered to the guidelines of the Pigs were sedated by an intravenous injection of sodium pentobarbital and then euthanized by electrocution as previously described (Beaver et al., 2001).Lung, liver, tonsil, kidney, spleen, small and large intestine and superficial inguinal lymph node tissues were collected from each pig at the time of necropsy.Tissues were fixed for 24 h in 10% neutral buffered formalin, routinely processed and embedded in paraffin.

Clinical observations
The pigs were monitored daily for abnormal clinical signs and scored weekly using scores ranging from 0 (normal) to 6 (severe dyspnea and abdominal breathing) (Halbur et al., 1995).Observers were blinded to vaccination and type of vaccine status.Mortality rate was calculated as the number of pigs that died divided by the number of pigs initially assigned to that group within batch.Pigs that died or were culled throughout the study were necropsied.Evaluation of injection site reaction, including palpation, was performed 24 h post-vaccination.

Average daily weight gain
The ADWG during the different production stages was calculated as the difference between the starting and final weight divided by the duration of the stage.Data for dead or removed pigs were included in the calculation.

Quantification of PCV2d DNA
DNA will be extracted from serum, tissue, nasal and faecal samples using the commercial kit (QIAamp DNA Mini Kit, QIAGEN) to quantify PCV2d genomic DNA copy numbers by real-time PCR (Jeong et al., 2015).

Quantification of M. hyopneumoniae DNA in larynx
DNA was extracted from laryngeal swabs using the commercial kit (QIAamp DNA Mini Kit, QIAGEN) to quantify the M. hyopneumoniae genomic DNA copy numbers by real-time PCR (Dubosson et al., 2004).

Serology
The serum samples were tested using the commercially available PCV2 (INgezim CIRCO IgG, Ingenasa) and M. hyopneumoniae (M.hyo.Ab test, IDEXX Laboratories Inc.).Samples were considered positive for PCV2 antibodies if the sample-to-positive (S/P) ratio was ≥0.3 and for M. hyopneumoniae antibody if S/P ratio was ≥0.4 in accordance with the manufacturer's instructions for each kit.The serum samples were tested using serum virus neutralization test against PCV2d (Fort et al., 2009;Pogranichnyy et al., 2000;Shen et al., 2010).

Enzyme-linked immunospot assay
Enzyme-linked immunospot (ELISPOT) assay was conducted to measure the numbers of PCV2d-specific and M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC) (Jeong et al., 2015(Jeong et al., , 2018)).The numbers of PCV2d-and M. hyopneumoniae-specific IFN-γ-SC were determined in peripheral blood mononuclear cells (PBMCs).The IFN-γ positive spots on the membranes were imaged, analysed and counted using an automated ELISPOT Reader (AID ELISPOT Reader, AID GmbH).The results were expressed as the numbers of IFN-γ-SC per million PBMC.ELISPOT assay was done in duplicate.

Pathology
The severity of macroscopic lung lesions was scored to estimate the percentage of the lung affected by pneumonia.The scoring was done by two pathologists (Chae and one graduate student) at the Seoul National University (Seoul, Republic of Korea).For the entire lung, 100 points were be assigned as follows: 10 points each to the right cranial lobe, right middle lobe, left cranial lobe and left middle lobe, 27.5 points each to the right caudal lobe and left caudal lobe and 5 points to the accessory lobe (Halbur et al., 1995).Two veterinary pathologists then examined the collected lung and lymphoid tissue sections and scored the severity of peribronchiolar and perivascular lymphoid tissue hyperplasia by mycoplasmal pneumonia lesions (0-6) (Opriessnig et al., 2004).Lymphoid lesion severity was scored (0-5) based on lymphoid depletion and granulomatous inflammation (Kim & Chae, 2004).

Statistical analysis
Real

Clinical signs
Vaccinated animals within farm A were significantly (p < 0.05) lower in

Average daily weight gain
A difference in mean body weight was not observed between vaccinated and unvaccinated animals at the time of vaccination on all three farms.In farms A and B, vaccinated animals were significantly (p < 0.05)

Mortality
Mortality at all farms was primarily related to infection with PCV2d
28, 49, 91 and 154 dpv.In farm C, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in their blood compared to those of unvaccinated animals at 28 and 49 dpv (Figure 2a).
In farms A and C, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in nasal samples compared to those of unvaccinated animals at 28, 49 and 91 dpv.In farm B, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in their blood compared to those of unvaccinated animals at 28 and 49 dpv (Figure 2b).
In farms A and C, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in oral samples compared to those of unvaccinated animals at 28, 49 and 91 dpv.In farm B, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in their blood compared to those of unvaccinated animals at 28, 49, 91 and 154 dpv (Figure 2c).
In farms A and B, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in lung tissues compared to those of unvaccinated animals at 91 dpv.In farm A, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in lung tissues compared to those of unvaccinated animals at 154 dpv (Table 1).
In farms A, B and C, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in tonsillar and lymph node tissues compared to those of unvaccinated animals at 91 dpv.In farm A, vaccinated animals had a significantly (p < 0.05) lower amount of PCV2d genomic copies in lymph node tissues compared to those of unvaccinated animals at 154 dpv (Table 1).

Immune responses against PCV2
Immune responses against PCV2 were consistent in animals across the evaluated three farms during this field trial.Vaccination of animals significantly (p < 0.05) increased the levels of PCV2 antibody (Figure 4a), neutralizing PCV2d antibody (Figure 4b) and PCV2d-specific IFN-γ-SC (Figure 4c) compared to unvaccinated animals at 28, 49, 91 and 154 dpv.

Immune responses against M. hyopneumoniae
Immune responses against M. hyopneumoniae were consistent in animals across the evaluated three farms during this field trial.Vaccination of animals significantly (p < 0.05) increased the levels of M. hyopneumoniae antibody (Figure 5a) and M. hyopneumoniae-specific IFN-γ-SC (Figure 5b) compared to unvaccinated animals at 28, 49, 91 and 154 dpv.

DISCUSSION
The evaluated field trial conferred protection against subclinical PCV2d infection and enzootic pneumonia in pigs that received a The evaluated bivalent vaccine containing PCV2d and M. hyopneumoniae elicited immune responses against field strains of PCV2d and M. hyopneumoniae, even though the precise protective immunity is not well known yet.PCV2d protection is often measured by level of neutralizing antibodies and IFN-γ-SC, both of which are well-known immune responses that are responsible for the clearance of PCV2 in the blood (Fort et al., 2012;Martelli et al., 2011;Meerts et al., 2005Meerts et al., , 2006)).Bivalent vaccination of pigs induced a high level of neutralizing anti-PCV2d antibodies and PCV2d-specific IFN-γ-SC which resulted in the reduction of both PCV2d blood viral load and lymphoid lesion severity.All of these, in turn, have had a positive effect on pig growth performance.
The bivalent PCV2d and M. hyopneumoniae vaccine evaluated in this study reduced PCV2d load in both the nasal and faecal samples under field conditions to similar levels of previous experimental studies (Patterson et al., 2011).PCV2 reduction of nasal and faecal shedding is a clinically meaningful comparison, as the nasal route may be more effective in transmission than through the faecal route (Patterson et al., 2011).Transmission through nasal and faecal secretions has been suggested as a potential mode of horizontal spreading (Patterson et al., 2011).Vaccination would be an effective disease control tool by lowering the risk of transmission to other pigs by decreasing the amount of PCV2 circulating within the herd.Pig tonsil tissue samples that produced a mean concentration between 10 4 and 10 6 PCV2 genome copies/ng of total DNA were considered subclinically infected (Segalés Calsamiglia et al., 2005).The PCV2 mean concentrations from tonsillar tissue per mL ranged from 0.56 × 10 1 to 5 × 10 1 PCV2 genomic copies/ng of total DNA in vacci-nated animals and from 2. Humoral immunity has not been associated with M. hyopneumoniae protection (Djordjevic et al., 1997), but cell-mediated immunity has been previously correlated (Thacker et al., 2000).Bivalent vaccination induced high levels of M. hyopneumoniae-specific IFN-γ-SC that resulted in the reduction of mycoplasmal laryngeal loads and reduced the severity of mycoplasmal lung lesions, all of which contributed to improve pig growth performance.
Mycoplasma quantification in the lungs of live, naturally infected pigs, is an appropriate evaluation of disease, as M. hyopneumoniae infects the lower respiratory tract, although quantitative analysis may be limited.Laryngeal swabs offer a more practical alternative for collecting samples for M. hyopneumoniae analysis under field conditions.
Laryngeal swabs were successfully used as a reliable sampling method for the early detection of M. hyopneumoniae, followed by bronchoalveolar lavage fluid and nasal swabs in live experimentally infected pigs, particularly during the acute period of infection (Pieters et al., 2017).
Optimal vaccination timing is critical in efficiently controlling PCV2 infection, yet this is a difficult process.and/or PCV2b (Um et al., 2022;Yang et al., 2020Yang et al., , 2021) ) although PCV2d is the most prevalent PCV2 genotype currently circulating throughout Asia (Dinh et al., 2021;Park & Chae, 2021;Thangthamniyom et al., 2017;Tsai et al., 2019;Yang et al., 2018).It has been also reported that PCV2d is more virulent than PCV2a or PCV2b (Oh et al., 2021).The efficacy of PCV2 vaccination may depend on the PCV2 field viruses that are in circulation when pigs are naturally exposed to the disease (Takahagi et al., 2010).An additional comparative field live weight of each pig was measured at 21 (0 dpv), 70 (49 dpv), 112 (91 dpv) and 175 (154 dpv) days of age.The average daily weight gain (ADWG; grams/pig/day) was analysed over three time periods: (i) between 21 and 70 days old, (ii) between 70 and 112 days old, (iii) between 112 and 175 days old and (iv) between 21 and 175 days old.

F
I G U R E 1 Mean respiratory score from vaccinated and unvaccinated groups in farms A, B and C. Variation is expressed as the standard deviation.*Significant difference (p < 0.05) between vaccinated and unvaccinated group within the same farm.
respiratory clinical signs compared to unvaccinated animals at14, 21,   42, 56, 91, 105, 112, 126  and 133 dpv.Respiratory clinical signs of farm B vaccinated animals were significantly (p < 0.05) lower compared to those of unvaccinated animals from 21 to 70 dpv.Respiratory clinical signs of farm C vaccinated animals were significantly (p < 0.05) lower compared to those of unvaccinated animals from 14 to 49 and at 98, 105 and 126 dpv (Figure 1).
and M. hyopneumoniae in unvaccinated animals.One 77-day-old vaccinated pig from farm A died of unknown acute haemorrhagic enteritis, one 68-day-old unvaccinated pig died of enzootic pneumonia caused by a co-infection with M. hyopneumoniae and Pasteurella multocida, and one unvaccinated 95-day-old pig died of enzootic pneumonia caused by a co-infection with M. hyopneumoniae and Trueperella pyogenes.One 88-day-old unvaccinated pig from farm B died of enzootic pneumonia caused by M. hyopneumoniae and T. pyogenes.Two vaccinated pigs died during the study in farm C; one at 55-day-old from streptococcal meningitis, and one 76-day-old from Glasser's disease caused by Glaesserella parasuis infection.Three unvaccinated pigs also died in farm C during the study; one 54-day-old from pneumonia caused by a co-infection with PCV2d and Staphylococcus aureus, and two from pneumonia caused by co-infection with PCV2d, M. hyopneumoniae and P. multocida at 77 and 82 days of age.

F
Mean values of the genomic copy number of porcine circovirus type 2d (PCV2d) DNA in serum (a), nasal (b) and faecal (c) samples of pigs from vaccinated and unvaccinated groups in farms A, B and C. Variation is expressed as the standard deviation.*Significant difference (p < 0.05) between vaccinated and unvaccinated group within the same farm.3.5 Quantification of M. hyopneumoniae DNA Vaccinated animals from farms A and C had significantly (p < 0.05) lower amounts of laryngeal M. hyopneumoniae genomic copies com-pared to those of unvaccinated animals at 49, 91 and 154 dpv.Vaccinated animals from farm B had significantly (p < 0.05) lower amounts of laryngeal M. hyopneumoniae genomic copies compared to those of unvaccinated animals at 91 and 154 dpv (Figure 3).

F
Mean values of the genomic copy number of Mycoplasma hyopneumoniae DNA in larynx of pigs from vaccinated and unvaccinated groups in farms A, B and C. Variation is expressed as the standard deviation.*Significant difference (p < 0.05) between vaccinated and unvaccinated group within the same farm.
farms A, B and C, vaccination of animals significantly (p < 0.05) reduced the severity of macroscopic and microscopic lung lesions and microscopic lymphoid lesions (Figure 6a,b) compared to unvaccinated animals at 91 (112 days of age) and 154 (175 days of age) dpv.Vaccination of animals significantly (p < 0.05) reduced the number of PCV2 antigen-positive cells in lymph nodes compared to unvaccinated animals at 91 (112 days of age), and 154 (175 days of age) dpv (Table PCV2d and M. hyopneumoniae bivalent vaccine.The most common clinical characteristic of both subclinical PCV2 infection and enzootic pneumonia is growth retardation of the pigs.As a result, growth performance was considered the critical index for successfully evaluating the efficacy of a bivalent vaccine under field conditions.Vaccination of pigs with the evaluated bivalent vaccine improved the growth performance in all three swine herds suffering from subclinical PCV2d infection and enzootic pneumonia under field conditions.Commercially, raised pigs are continuously exposed and re-exposed to PCV2d field strains throughout their production process, which is why the positive effect of vaccination on growth performance demonstrated in this field trial is clinically significant.

F
Mean values of the porcine circovirus type 2 (PCV2) ELISA titres (a), the PCV2d-specific neutralizing antibodies (b) and PCV2d-specific IFN-γ-SC/10 6 peripheral blood mononuclear cell (PBMC) (c) from vaccinated and unvaccinated groups in farms A, B and C. Variation is expressed as the standard deviation.*Significant difference (p < 0.05) between vaccinated and unvaccinated group within the same farm.
7 × 10 4 to 9.4 × 10 5 PCV2 genomic copies/ng of total DNA in unvaccinated animals from all three farms at 112 days of age (91 days post-vaccination) for the present study.This indicates that the bivalent vaccine evaluated in these field trials conferred protection against subclinical PCV2 infection.F I G U R E 5 Mean values of the Mycoplasma hyopneumoniae ELISA sample-to-positive (S/P) ratio (a) and M. hyopneumoniae-specific IFN-γ-SC/10 6 peripheral blood mononuclear cell (PBMC) (b) from vaccinated and unvaccinated groups in farms A, B and C. Variation is expressed as the standard deviation.*Significant difference (p < 0.05) between vaccinated and unvaccinated group within the same farm.
For example, vaccine failure may occur if piglets are vaccinated too early due to the prevalence of high maternal antibody.Maternally derived antibodies (MDA) may interfere with vaccine efficacy in 3-week-old pigs, which is the traditional timeframe for piglet vaccination.The present field trial demonstrated that the bivalent vaccine elicited systemic humoral and cellular immune responses to PCV2d and M. hyopneumoniae even in the presence of MDA.Therefore, an MDA effect on active immunization after vaccination at 3 weeks of age is less likely to hamper the efficacy of this bivalent vaccine.This study was the first field trial that evaluated a PCV2d and M. hyopneumoniae bivalent vaccine in multiple herds already suffering from subclinical PCV2d infection and enzootic pneumonia.This study was necessary as most of the commercially bivalent vaccines of PCV2 and M. hyopneumoniae available today are based on PCV2a

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I G U R E 6 Histopathology of vaccinated pig (a) and unvaccinated pig (b) from farm A. Vaccinated pig had mild lymphoid depletion (arrows) and unvaccinated pig had moderate granulomatous inflammation (arrows).trial that evaluated and determined that a trivalent vaccine containing PCV2a/b and M. hyopneumoniae was more effective in improving growth performance than a bivalent vaccine containing PCV2a and M. hyopneumoniae (Um et al., 2021) provides additional information in the broader PCV2 genomic picture.PCV2d-caused PCVAD has been reported in PCV2a-vaccinated herds (Opriessnig et al., 2013; Ramos et al., 2015; Seo et al., 2014).Despite these occasional instances of vaccine failure, several similar commercial bivalent vaccines made up of various PCV2 genotype antigens are used globally and have proven as efficacious in controlling co-infections with PCV2d and M. hyopneumoniae.Additional direct comparison studies are needed to determine efficacy differences among these bivalent vaccines containing different PCV2 genotype antigens in farms suffering from co-circulating PCV2d and M. hyopneumoniae.
Average daily weight gain (ADWG), body weight, mortality rate and pathology between vaccinated (Vac) and unvaccinated (UnVac) animals on three farms.
TA B L E 1